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1.
为建立一种能够快速鉴别检测猪繁殖与呼吸综合征病毒(PRRSV) GDr180疫苗株与GD野毒株的双重荧光定量RT-PCR方法,本研究在PPRSV的Nsp2基因和Orf7基因区域分别设计不同荧光标记的MGB探针及特异性引物.这两种MGB探针能够分别特异结合GD野毒株和GDr180疫苗株.通过对PRRSV培养液、感染猪血清和组织样品检测证明了该方法的可行性;采用双重实时荧光定量RT-PCR方法检测PRRSV GDr180疫苗株和GD强毒株时敏感性分别达到19.4拷贝/μL和34.2拷贝/μL;该方法与PRRSV其他8个病毒株和猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型和猪细小病毒不发生交叉反应,表明该方法具有良好的特异性;因此可以用于PRRSV GDr180疫苗株与野毒株的鉴别检测. 相似文献
2.
为了解猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)强、弱毒株在PAM细胞上的增殖特性,本研究在体外分离培养了健康猪肺泡巨噬细胞(porcine alveolar macrophages,PAM),然后分别用高致病性PRRSV强毒HuN4株和弱毒疫苗HuN4-F112株感染PAM细胞,细胞病变观察和间接免疫荧光检测结果显示,二者在体外均可以感染PAM细胞,其中强毒HuN4株感染PAM细胞CPE较为明显。在两个毒株感染PAM细胞后12、24、36、48、60h分别收获病毒感染的细胞,利用抗PRRSV N蛋白单抗进行Western blot分析检测病毒核蛋白在不同时间表达量的变化,结果表明,强毒株在感染PAM细胞的早期,N蛋白合成表达量明显高于弱毒疫苗株,而弱毒疫苗株在感染Macr-145细胞早期,N蛋白的合成量则明显优于强毒株。比较HuN4株与HuN4-F112株在PAM细胞和Marc-145细胞的生长曲线,结果显示强、弱毒在PAM细胞和Marc-145细胞生长趋势存在明显差异,其中强毒HuN4株在PAM细胞上增殖能力明显强于弱毒株,而弱毒HuN4-F112株在Marc-145细胞上的增殖能力明显强于其在PAM细胞上的增殖能力,表明PRRSV强毒株对靶细胞PAM的感染能力较强,弱毒疫苗株对其感染能力相对较弱。本研究为深入了解PRRSV强毒株与弱毒疫苗株的致病性差异提供了理论依据。 相似文献
3.
2013年初上海某猪场保育猪群中出现高热、呼吸障碍等临床表现,导致大部分发病猪死亡,为了确定此次猪群中爆发疫病的病原,本研究从发病猪群中随机采集了15份血液样品,利用RT-PCR方法对本次疫病病原进行了检测。结果显示,有11份临床样品呈现猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)强阳性,且与高致病性猪繁殖与呼吸综合征病毒(Highly pathogenic Porcine reproductive and respiratory syndrome virus,HP-PRRSV)对照大小相一致。随后从阳性样品中选取SHxx13/2013在Marc-145细胞中进行病毒的分离与鉴定,结果显示SHxx13/2013在细胞接种后第1代即出现明显的细胞效应(cytopathic effect,CPE),其病变特征与高致病性PRRSV强毒HuN4株一致。对SHxx13/2013第5代细胞分离毒进行RT-PCR和IFA等特异性鉴定,结果显示该毒株为PRRSV分离株,其蚀斑形态和生长特性与强毒HuN4株相似。分段克隆该毒株全长基因进行测序和序列分析,结果显示,新分离的流行毒SHxx13/2013株全长基因组为15 319 bp,其Nsp2基因特征与HP-PRRSV一致,在第482位和第534~562位发生两处共30个氨基酸的不连续缺失,且该毒株与高致病性PRRSV代表毒株HuN4亲缘关系较近,全长基因的核苷酸同源性为99.6%。本研究结果表明新分离的SHxx13/2013株属于高致病性PRRSV分离株,据此我们推测今年年初上海某猪场爆发的疫情主要是由HP-PRRSV感染所致。 相似文献
4.
为建立一种能够区分猪瘟病毒(CSFV)强毒与弱毒疫苗C株的SYBR Green Ⅰ荧光定量RT-PCR结合熔解曲线分析方法,本研究对GenBank中登录的25株CSFV强毒株和兔化弱毒疫苗C株全基因组序列进行比较分析,设计一对共用下游引物以及分别针对CSFV强毒株与弱毒疫苗的特异性上游引物,其Tm值分别为84.5±0.5℃和88.5±0.5℃,熔解曲线分析显示为单特异峰.检测结果显示本实验建立的鉴别CSFV强毒感染与弱毒疫苗的荧光定量RT-PCR结合熔解曲线分析方法特异性强,对其他相关病毒无特异性扩增;敏感性高,最低检出量为5×RID50的细胞疫苗基因组拷贝;重复性好;并且扩增效率高、线性范围广、检测时间短,可对免疫猪群中CSFV强毒感染做出快速准确的鉴别检测,为有效防制猪瘟提供依据. 相似文献
5.
中国农业大学动物医学院教授杨汉春作了题为《我国PRRS的过去、现在与未来》的第一个报告,杨教授在报告中指出,由于HP-PRRSV的出现、变异与演化,多个HP-PRRSV减毒活疫苗的无序、盲目、高频使用,PRRSV的重组,HP-RRSV减毒活疫苗的安全性降低与毒力返强,以及猪场疫苗毒与野毒的共存,使得PRRSV毒株多样性剧增,从而导致PRRSV的控制难度加大.另外,近年来,北美毒株NADC30的传入使中国养猪业雪上加霜:类NADC30毒株自2014年开始流行以来,已经成为我国猪场猪蓝耳病毒主要流行毒株之一,该毒株感染主要引起以母猪流产等繁殖障碍为特征,而且该毒株极易与我国毒株(包括野毒株和疫苗毒株)发生重组,毒力属于中等偏强,现有疫苗不能完全抵抗该毒株的感染. 相似文献
6.
《中国兽医学报》2016,(12)
为了比较国内不同猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)弱毒疫苗的安全性和有效性,本研究将17头PRRSV抗原和抗体双阴性的28日龄仔猪随机分为4组,TJM-F92株弱毒疫苗组,JXA1-R株弱毒疫苗组,经典株弱毒疫苗(VR2332来源)组和注射生理盐水对照组。免疫后7,14,21,28d分别采血,ELISA抗体检测试剂盒检测PRRSV抗体,结果从免疫后14d起PRRSV抗体开始转阳,但各试验组的抗体水平无明显差异。对免疫后14d所采血样进行病毒分离,TJM-F92株免疫组未分离到PRRSV疫苗毒,JXA1-R株免疫组2头猪分离到PRRSV疫苗毒,经典株免疫组只1头猪分离到PRRSV疫苗毒。免疫后28d,所有猪接种PRRSV强毒株TJ株进行攻毒试验,观察各组猪的体温、临床症状;攻毒后21d,所有猪安乐死,剖检观察肺部病理变化。结果显示,TJM-F92株和JXA1-R株免疫组均未出现临床发病猪,经典株免疫组2头猪出现临床发病,对照组全部临床发病,死亡1头。经典株免疫组临床发病猪出现典型PRRSV感染肺部病理变化,而TJM-F92株和JXA1-R株免疫组均未发现典型PRRSV感染肺部病理变化。本研究证实TJM-F92株和JXA1-R株对高致病性PRRSV的免疫效果优于经典株弱毒疫苗,TJM-F92疫苗株与JXA1-R疫苗株相比,疫苗毒在体内带毒时间更短,安全性更高。 相似文献
7.
为评价一种短肽佐剂对猪繁殖与呼吸综合征病毒(PRRSV) CH-1R弱毒疫苗的免疫增强效果,本研究分别采用CH-1R疫苗株单独(单独免疫组)、CH-1R疫苗株与短肽佐剂共同(共同免疫组)及生理盐水(对照组)免疫仔猪,4周后以高致病性PRRSV (HP-PRRSV)TJ-F5攻毒,并采用流式细胞术对攻毒前后淋巴细胞亚类T、Th、Tc及NK进行绝对和相对计数.结果显示攻毒后共同免疫组仅在第3d淋巴细胞亚类数量显著下降后回升,28 dTc/T显著高于单独免疫组,临床症状和各组织器官病理变化轻微;单独免疫组在攻毒后第3d淋巴细胞亚类数量显著下降后回升,之后于第14和15d再次显著下降后又回升,临床症状和各组织器官病理变化比共同免疫组严重,但比对照组明显减轻.单独免疫组、共同免疫组及对照组的攻毒保护率分别为40％、80％、0.研究结果表明,短肽佐剂配合CH-1R弱毒疫苗具有缓解HP-PRRSV T J-F5的免疫抑制、增强细胞免疫及提高免疫保护力的作用;在抵抗HP-PRRSVT J-F5攻击时,共同免疫组的免疫保护力明显高于单独免疫组. 相似文献
8.
猪繁殖与呼吸综合征病毒弱毒疫苗和变异株灭活疫苗的免疫效果评价 总被引：7，自引：1，他引：6
分别采用猪繁殖与呼吸综合征病毒(PRRSV)CH-1R致弱毒疫苗株和变异毒株灭活疫苗免疫接种40日龄～45日龄仔猪,免疫接种后4周,用高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)HuN-4变异株强毒攻毒,攻毒后21 d剖检,取主要器官作病理组织学观察和病毒抗原定位.结果显示变异毒株灭活疫苗免疫攻毒后导致的病理变化和病毒抗原分布程度均明显高于CH-1R弱毒疫苗免疫组.免疫病理学研究结果表明CH-1R 弱毒疫苗对HU-4株强毒免疫保护效果好于变异毒株灭活疫苗. 相似文献
9.
20日龄猪繁殖与呼吸综合征(PRRS)阴性健康仔猪滴鼻感染猪繁殖与呼吸综合征病毒(PRRSV) Hn-1/06强毒株和BJ-4弱毒株,于接种后0、5、11、18、30、40、50 d无菌采血,制备血清和分离白细胞,并在感染猪发病死亡或第50天后分别剖杀取各器官组织.经RT-PCR方法检测,强弱毒株在血清、白细胞和各组织器官内分布基本一致;经ELISA方法和免疫荧光抑制试验及IFA流式细胞术检测,强弱毒株感染均诱导机体产生抗N蛋白抗体,强毒株的抗N蛋白抗体产生早于弱毒株;强毒株感染未能诱导产生中和抗体,弱毒株感染后第30天诱导产生了较低水平的中和抗体,随后逐步升高;在整个感染过程中,强毒感染猪CD3+T淋巴细胞及CD4+和CD8+T淋巴细胞亚群均下降,CD4+/CD8+比值远远小于对照组,弱毒感染猪CD3+T淋巴细胞及CD4+和CD8+T淋巴细胞亚群先下降,40 d后逐渐恢复正常水平,CD4+/CD8+比值先下降,11 d后开始逐步上升,18 d后超过对照组,近50 d恢复到正常水平.结果说明,在强毒株感染过程中,细胞免疫被抑制,不能产生有效的体液免疫,导致病毒快速增殖复制;在弱毒株感染过程中,细胞免疫和体液免疫先被抑制,后免疫功能逐步恢复正常水平,使体内病毒进一步被清除. 相似文献
10.
2022年10月中旬，湖南长沙某规模化猪场发生疑似猪繁殖障碍综合征疫情。为探究发病原因，采集病料样品进行细菌分离鉴定和常见病原荧光定量PCR（q PCR）鉴定。结果发现，病料样品中未分离得到细菌，q PCR检测结果显示组织病料为HP-PRRSV和NADC30-like毒株核酸阳性，其他病原核酸均为阴性。为进一步分析该猪场PRRSV流行株遗传变异情况，对PRRSV Nsp2基因序列进行逆转录PCR（RT-PCR）扩增、测序及分析。结果显示，HP-PRRSV株Nsp2基因序列与Hu N4-F112株（PRRSV弱毒疫苗株）同源性最高，类NADC30株Nsp2基因序列与已知NADC30株同源性最高。以上研究结果表明，该场疫情是HP-PRRSV和类NADC30株混合感染引起，其中HP-PRRSV可能为弱毒疫苗株。 相似文献
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12.
为研究猪霍乱沙门氏菌C79102株的菌种特性,制备了一批猪霍乱沙门氏菌C79102株,并对菌种的真空度、纯粹、剩余水分、培养特性、形态及生化特性、血清学特性、特异性、毒力等进行检定,重点对菌种的毒力进行研究。试验结果表明,该冻干菌种的真空度、纯粹、剩余水分、培养特性、形态及生化特性、血清学特性均符合《中华人民共和国兽用生物制品规程》二〇〇〇版质量标准的规定,特异性检验结果显示C79102在含1%醋酸铊的普通肉汤中不生长,毒力试验结果显示,不同制备方法获得的肉肝胃消化汤培养的菌液毒力差异明显。本研究可以为猪霍乱沙门氏菌的制备和检定提供参考依据。 相似文献
13.
鹅细小病毒灭活疫苗种子批建立 总被引：1，自引：1，他引：0
为保证制苗所用毒种质量和稳定性,在对制苗毒种病毒含量、病毒纯净性、特异性、免疫原性及扩繁代次等研究的基础上,建立了鹅细小病毒灭活疫苗毒种种子批。试验使用鹅胚对鹅细小病毒YA98株进行了20次传代。传代毒种的鉴定结果表明:抽检的各代次的毒种均无细菌、支原体、外源病毒污染,且病毒含量检测稳定,每0.3 m L含104.78~4.85ELD50;各代次特异性检测均能被鹅细小病毒抗血清中和;将各代次病毒液制成灭活疫苗,免疫成鹅后均能产生完全保护。以此为依据,最终确定原始毒种和基础毒种的扩繁代次宜控制在5代以内,生产毒种的最高扩繁代次宜控制在15代以内。种子批的建立,为鹅细小病毒灭活疫苗的生产奠定了基础。 相似文献
14.
15.
Sri Hartati Asmarani Kusumawati Hastari Wuryastuti J. Sri Widada 《Journal of veterinary science (Suw?n-si, Korea)》2006,7(3):263-270
Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1. 相似文献
16.
Alexandre Olvera Maria Ballester Miquel Nofrar��as Marina Sibila Virginia Aragon 《Veterinary research》2009,40(3)
Haemophilus parasuis is a colonizer of the upper respiratory tract of healthy pigs, but virulent strains can cause a systemic infection characterized by fibrinous polyserositis, commonly known as Glässer’s disease. The variability in virulence that is observed among H. parasuis strains is not completely understood, since the virulence mechanisms of H. parasuis are largely unknown. In the course of infection, H. parasuis has to survive the host pulmonary defences, which include alveolar macrophages, to produce disease. Using strains from different clinical backgrounds, we were able to detect clear differences in susceptibility to phagocytosis. Strains isolated from the nose of healthy animals were efficiently phagocytosed by porcine alveolar macrophages (PAM), while strains isolated from systemic lesions were resistant to this interaction. Phagocytosis of susceptible strains proceeded through mechanisms independent of a specific receptor, which involved actin filaments and microtubules. In all the systemic strains tested in this study, we observed a distinct capsule after interaction with PAM, indicating a role of this surface structure in phagocytosis resistance. However, additional mechanisms of resistance to phagocytosis should be explored, since we detected different effects of microtubule inhibition among systemic strains. 相似文献
17.
小反刍兽疫病毒属于副黏病毒科麻疹病毒属,为有囊膜的单股负链RNA病毒.以灭活的检测用抗原为材料,抽提基因组RNA作为模板,根据GenBank下载的序列及进行原核表达载体的要求,设计可扩增小反刍兽疫病毒N、M、F及H 4个基因的全长读码框(ORF)的引物,进行RT-PCR扩增及扩增片段的T载体克隆、序列分析.结果显示,均有预期大小的片段扩增出.扩增片段经核苷酸序列测定、分析,N、M、F及H基因ORF全长分别为1 578、1 008、1 641、1 830 nt; 4个基因均与疫苗株Nigeria 75/1(X74443)有100%的同源性,说明所购试剂盒用的检测抗原应该是用此疫苗株制备,也证明了设计用于原核表达的4对引物扩增片段的正确性. 相似文献
18.
Pengcheng LI Yunfeng LI Guoqing SHAO Qinghua YU Qian YANG 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2015,77(5):519-525
The aim of this study was to evaluate the immune responses to intranasal and
intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae
(Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four
pigs were randomly divided into 4 groups: an intranasal immunization group, an
intrapulmonary immunization group, an intramuscular immunization group and a control
group. The levels of local respiratory tract cellular and humoral immune responses were
investigated. The levels of interleukin (IL)-6 in the early stage of immunization
(P<0.01), local specific secretory IgA (sIgA) in nasal swab samples
(P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and
trachea were higher after intranasal vaccination (P<0.01) than in the
control group. Interestingly, intrapulmonary immunization induced much stronger immune
responses than intranasal immunization. Intrapulmonary immunization also significantly
increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and
IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and
the numbers of CD4+ and CD8+ T lymphocytes in the lung and hilar
lymph nodes were significantly increased by intrapulmonary immunization compared with
those in the control group (P<0.01). These data suggest that
intrapulmonary immunization with attenuated Mhp is effective in evoking
local cellular and humoral immune responses in the respiratory tract. Intrapulmonary
immunization with Mhp may be a promising route for defense against
Mhp in pigs. 相似文献
19.
Daisuke TAKAMATSU Masumi SATO Mikio YOSHIYAMA 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2016,78(1):29-34
Melissococcus plutonius is an important pathogen that causes
European foulbrood (EFB) in honeybee larvae. Recently, we discovered a group of M.
plutonius strains that are phenotypically and genetically distinct from other
strains. These strains belong to clonal complex (CC) 12, as determined by multilocus
sequence typing analysis, and show atypical cultural and biochemical characteristics
in vitro compared with strains of other CCs tested. Although EFB is
considered to be a purely intestinal infection according to early studies, it is unknown
whether the recently found CC12 strains cause EFB by the same pathomechanism. In this
study, to obtain a better understanding of EFB, we infected European honeybee
(Apis mellifera) larvae per os with a
well-characterized CC12 strain, DAT561, and analyzed the larvae histopathologically.
Ingested DAT561 was mainly localized in the midgut lumen surrounded by the peritrophic
matrix (PM) in the larvae. In badly affected larvae, the PM and midgut epithelial cells
degenerated, and some bacterial cells were detected outside of the midgut. However, they
did not proliferate in the deep tissues actively. By immunohistochemical analysis, the PM
was stained with anti-M. plutonius serum in most of the DAT561-infected
larvae. In some larvae, luminal surfaces of the PM were more strongly stained than the
inside. These results suggest that infection of CC12 strain in honeybee larvae is
essentially confined to the intestine. Moreover, our results imply the presence of
M. plutonius-derived substances diffusing into the larval tissues in
the course of infection. 相似文献